The term “retroviral vectors” or “retroviral transfervectors” refers to infectious but replication incompetent retroviruses, which are able to transfer genes as retroviral expression constructs (also designated as “expression vectors”) into cells. The gene transfer results in the integration of the expression construct into the genome of the cell. Retroviral gene transfer is advantageous, because (1) usually one copy of the gene of interest is transferred into cells, (ii) the gene generally is transferred without any mutations or recombination and (iii) stable chromosomal integration occurs.
It is known to use retroviral vectors derived from the amphotropic murine leukemia virus (MLV) to transfer certain genes into mammalian cells, especially human cells. These vectors are replication incompetent and run only through one cycle of replication. For the preparation of such vectors two components are required. On the one hand, a packaging cell is required, that provides the gag-, pol- and env-gene products of MLV upon expression of psi-negative constructs so that these genes can not be packaged into a retrovirus. “psi” designates the packaging signal of retroviruses, that mediates efficient packaging of messenger RNA. On the other hand, a so called expression construct has to be generated, that allows packaging into the retroviral vector and transfer by the retrovirus and that encompasses a coding and translatable region of the desired gene product. Thus, the expression construct has to contain the packaging signal “psi”. The genes gag-, pol and env within the untreated packaging cell must be psi-negative to prevent the respective messenger RNA from being packaged into retroviral particles. Upon transfer of the expression construct by transfection of the respective vector-DNA into the packaging cells, retroviral vector particles are released into the cell culture supernatant, that exclusively contain the expression construct, but not the psi-negative gag-, pol and env genes, which are thus not transferred into the target cells.
The tropism of retroviral vectors, i.e. the selection of mammalian cells into which these retroviral vectors can transfer the expression construct, is determined by the env gene in the respective packaging cell. The env gene is translated into envelope proteins; the transmembrane protein (TM) and the surface envelope protein (SU), which together form the outer envelope of the retroviral vector particle. The env gene products of the amphotropic MLV, which is widely used for gene transfer, mediate gene transfer into a variety of different mammalian cells. However, in particular for gene transfer into human cells the amphotropic retroviral vector does not allow specific gene transfer into selected human or other mammalian tissues or cell species, as the acceptor protein (receptor) for the amphotropic MLV-envelope proteins which mediates the uptake of amphotropic retroviral vector particles, and the gene transfer is found on the surface of almost all mammalian cells.
In gene therapy, today, stable transfer of different genes is mostly performed in cell culture, i.e. “ex vivo”. Retroviral vectors were improved by exchanging the retroviral env gene of MLV within the packaging cells by env genes derived from other viruses. As an example the env gene of MLV has been exchanged by the env gene encoding the protein G of “vesicular stomatitis virus (VSV)” (Burns et al., Proc. Natl Acad. Sci. USA 90 (1993), 8033-8037). The resulting retroviral vectors are characterized by enhanced stability. In WO 96/17071 retroviral vectors are described, that harbor the env gene of “human spuma retrovirus” (HSRV) instead of the MLV env gene. As amphotropic MLV, HSRV does not reveal any specificity for the infection of cells. All mammalian cells that have been tested yet, are permissive for HSRV, independent from which donor species or tissue type they have been derived from (Schweizer et al., J. Virol 71 (1997), 4821-4824, and Russel et al, J. Virol. 70 (1996), 217-222). The possible use of the env genes of “simian sarcoma associated virus” (Takeuchi et al., Virology 186 (1992), 792-794), of the “feline leukemia virus subgroup B” (Porter et al., Hum. Gene Ther. 7 (1996), 913-919), of the “feline endogenous virus RD 14” (Cosset et al., J. Virol. 69 (1995), 7430-7436) and of the “human T-cell leukemia virus I (HTLV-I)” (Vile et al., Virology 180 (1991), 420-424) is suggested in certain experiments. Attempts to prepare retroviral vectors containing the env genes of the lentiviruses HIV-1, HIV-2 or “simian immunodeficiency virus (SIV)” have not been successful, yet. Such retroviral vectors would contain the capsid proteins encoded by the gag gene of MLV and the envelope proteins, encoded by the env gene of other retroviruses like HIV or SIV.
Vectors for the specific gene transfer into CD4-positive mammalian cells do not exist, yet.